In many monoclonal antibody (mAb) purification platforms, traditional anion exchange column chromatography or, increasingly, anion exchange membrane chromatography, is used as a polishing step in a product flowthrough mode to bind trace levels of process- or product-related impurities and assure efficient viral clearance. Anion exchange chromatography is, however, limited by the requirement for low loading buffer conductivity to efficiently remove impurities, which necessitates buffer exchange or dilution of the protein A column eluate. In this study, the authors developed a mAb polishing step using salt tolerant interaction membrane chromatography. Using a 96-well high-throughput screening (HTS) approach the authors identified the initial chromatographic parameters for acceptable step recovery and product quality. The authors then confirmed these conditions using small STIC capsules. Using a combination of HTS screening and design of experiments optimization the authors developed a mAb polishing platform which demonstrated high step recovery and efficient clearance of impurities (i.e., host cell proteins, high molecular weight species, host DNA, and leached protein A) for multiple antibodies at higher loading buffer conductivity. This simple and efficient polishing step can be easily integrated into most current mAb purification platforms, which may shorten mAb purification processes and accelerate development programs.

Monoclonal antibody (mAb) purification processes exist in different well-established platforms with extensive process performance histories for production of commercial monoclonal antibodies (1–10). These platforms, typically employing two or three chromatographic steps, are scalable and robust, and produce proteins with acceptable process yield and product quality.

In most of the two-column downstream processing platforms, the first chromatographic unit operation is protein A which binds the target mAb product directly from the harvested cell culture fluid (3, 4, 10–12). The process impurities are removed in the flowthrough and subsequent wash steps. A low pH buffer elutes the product and sets up the subsequent viral inactivation step. Anion exchange chromatography (AEX), such as Q Sepharose Fast Flow (Q FF) column chromatography (3, 13, 14) and Q membrane adsorber (6, 15–18), serves as the second chromatographic purification step. It is operated in a flowthrough mode, binding trace impurities such as host cell proteins, host DNA, endotoxins, and in some instances, high molecular weight (HMW) species while the antibody passes through. The AEX chromatography step is limited by the requirement for low loading buffer conductivity, which necessitates buffer exchange through tangential flow filtration (TFF) or dilution of the protein A column elution pool for efficient impurity clearance. However, some antibodies may have solubility issues at low ionic strength conditions. These challenges may be addressed by Sartorius Sartobind salt tolerant interaction chromatography (STIC) using a polyallylamine ligand covalently coupled to the double-porous membrane (19). The optimized base support membrane matrix combined with weak anion exchange chemistry provides a robust method for viral clearance at physiological conductivities and above (19, 20). A virus, ΦX174, used to model weak acidic contaminants, was shown to be removed (LRV >5) in the presence of 150 mM NaCl. Megta et al. demonstrated efficient viral clearance on STIC using two model viruses, MMV and MuLV (21). Furthermore, similarly to Q membrane chromatography, the STIC membrane adsorber may also provide some economic benefits as an alternative mAb polishing step (16, 22).

In this study, Sartobind STIC was evaluated as a mAb polishing platform alternative to Q column chromatography or Q membrane adsorber. Using a combination of high-throughput screening (HTS) and design of experiments (DOE) optimization, we developed a STIC mAb polishing platform which demonstrated high step recovery and efficient clearance of impurities (host cell proteins, host DNA, and leached protein A) for four antibodies at higher loading buffer conductivity. In addition, since there is no need for buffer exchange, the pre-Q column TFF step can be removed from the purification process. This polishing step, which can be easily integrated into current mAb purification platforms, offers a viable alternative to traditional AEX especially in cases where antibodies exhibit poor process performance. Furthermore, methods described here for developing STIC operating conditions can be applied to the purification process development of other membrane adsorbers.